This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Tuberculosis continues to pose a serious threat to human health with approximately 2 million deaths per annum. Our knowledge of mycobacterial pathogenesis is severely limited by the difficulties studying M. tuberculosis. Other pathogenic species, like Mycobacterium marinum, have been utilized to investigate pathogenic mechanisms of mycobacteria. Mycobacterium marinum causes a chronic, wasting disease in poikilotherms, and also is an opportunistic pathogen of humans. Several models have been developed to study M. marinum infection including: goldfish, leopard frogs, and zebrafish. The pathogenic mechanisms used by M. marinum are similar to those used by M. tuberculosis. Our goal is to identify new virulence determinants using a modified signature-tagged mutagenesis protocol in concert with the zebrafish model of mycobacterial pathogenesis. Instead of using tags that differ in sequence, we will use tags that differ in size. This will allow us to screen mutant libraries using a PCR methodology instead of using hybridizations to detect tags. The specific aims of the current proposal are: 1) construct signature-tagged mutants with different sized tags, 2) identify attenuated mutants, and 3) determine virulence genes in M. marinum. We hope to identify new virulence factors that will aid our understanding of mycobacterial pathogenesis, and lead to the development of new chemotherapeutics or vaccines against pathogenic mycobacteria.